Numerous xenobiotics as well as endobiotics such as prostaglandin, fatty acids and steroids are metabolized by mixed function oxidase (MFO) systems. The system consists of three components: cytochrome P-450 (P-450), NADPH-P-450-reductase and phospholipids. Cytochrome b5 (b5) is an additional electron carrier. P-450 is a key component of the MFO system and substrate specificity is directly associated with individual P-450 isozymes. Monoclonal antibodies (MAbs) were prepared to P450 isozymes, b5 and reductase in order to identify and characterize each component of MFO systems in tissues and organs and to understand their role in differential xenobiotic metabolism and sensitivity to chemical carcinogens. MAbs to P-450 2c/RLM5 were found to be regiospecific in steroid metabolism. Hybridization between myeloma cells and spleen cells derived from mice immunized with b5 yielded 29 independent hybrid clones which produced either IgGl(K), IgG2b(K), IgG3(K) or IgM (K) type MAbs. Out of 29 hybrids, 15 clones produced MAbs which were inhibitory up to 77% on b5-reductase activity. Eleven MAbs were specifically bound to a liver microsomal protein which corresponded to the chemically purified b5 on Western blots. These MAbs would be very useful for identification of each component of MFO systems in tissues and organs and analysis of their roles in MFO systems.